Enteric disease vaccine

ABSTRACT

Preparation of and live bacterial vaccine from selected strains of Escherichia coli. The bacterial cells are treated with dilute formalin solutions (about 0.02 to about 0.08% v/v formalin) for a period to modify the bacteria without inactivation thereof. One preferred strain is EW1 serogroup of 0157. The vaccine has been found particularly effective in preventing the occurrence of enteric colibacillosis in newborn animals. A preferred technique is to vaccinate cows, recover the milk, and continually feed it as a milk replacer to the newborn during the susceptible period.

United States Patent [1 1 Wilson Sept. 23, 1975 ENTERIC DISEASE VACCINE [75] Inventor: Michael R. Wilson, Guelph, Canada [73] Assignee: Canadian'Patents and Development Limited, Ottawa, Canada .[22] Filed: May 25, 1972 [21] Appl. No.: 256,977

[52] US. Cl 424/92; 424/87 [51] Int. Cl A6lk 23/00 [58] Field of Search 424/92 [56] References Cited OTHER PUBLICATIONS Wilson et al., Am. J. Vet. Res., 32(6): 891-898, June 1971, Immunity to Escherichia Coli in PigszSerologk Response of Sons Given Formalin-Treated Live Escherichia Coli Vaccine.

Svendsen et al., Am. .1. Vet. Res. 32(6): 899-904, June 1971, Immunity to Escherichia Coli in PigszEffect of Feeding Colostrum or Serum From Vaccinated Sows to Escherichia Coli-Infected Onotobiotic Pigs. Kohler et al., Am. J. Vet. Res. 29(7): 1419-1428, July 1968, Serological Response of Swine to Escherichia Coli Antigens.

Primary Examiner-Shep K. Rose Attorney, Agent, or FirmAlan A. Thomson 57 2 ABSTRACT 6 Claims, No Drawings 1 ENTERIC DISEASE VACCINE shown (control or non-vaccinated animals were not" kept in parallel). Well-controlled field trials with many vaccines gave no indication of protection. Inconsistent results with E. coli vaccines and the immunological treatment of E. coli associated enteritis have caused confusion.

Vaccines have been prepared from E. coli by growth on agar surfaces and washing off the live cells (the vaccine is free of culture and growth byproducts). Use of these active cells as vaccinesis believed to carry many risks e.g. infection, severe reaction, possible abortion etc. Formaldehyde has been used to inactivate bacterial cultures but the amount of formaldehyde and severity of treatment has been sufficient to kill the cells and to substantially alter the cell constituents. The anti- It has now been found according to this invention that a relatively safe and effective vaccine for coliform enteritis and related infections can be prepared by a controlled formalinization (or incomplete formaldehyde treatment) of selected E. coli strains. A broth culture of the E. coli bacteria is incubated for about 2 to about 20 hours in'the presence of about 0.02 to about 0.08% (volj/vol.) formalin (0.008 to 0.032% wt./vol.) formaldehyde). The cell and colony growth are obvious'ly modified (change in appearance, growth pattern and antigenic effect) yet many remain viable and are able to gradually recover their normal growth.

The vaccine normally contains the entire broth culture constituents including metobolic" waste products and extracellular proteins. During incubation with formalin the culture concentration is suitably-about 10 to about 10 viable organisms per ml. the count decreasing on incubation. The formalinization reduces the number of colony-forming units to approximately 3 log dilutions or more (e.g. 2-7) lower than are in the starting broth culture. This incubated broth is thenusually administered without-'furtherf dilution as the vaccine with at least about 10 viable organisms per ml.

The effect of yarying concentrations of formalin is illustrated in Example l. Below about 0.02% v/vformalin, the bacteria'were not significantly affected, and

above about 0.07 0.08% all were dead. About 0.04%

benefit is not achieved. For the shorter times it is preferred to use the higher formalin concentrations, with 0.030.057( formalin for 10-15 hours being very suitable.

The selected E. coli strain is grown in a standard broth culture medium under aerobic conditions. Tryptic soy broth has been found satisfactory but other liquid media such as peptone broth. a supplemented yeast extract etc. may be used. (Suitable growth media for E. coli are known in the art). The bacteria may be maintained on solid media and the cells removed and'suspended in a broth medium for the incubation.

One preferred strain :of E. coli is EWl serotype 0l5 7:KV17 which has been found to be very susceptible to formalin andto give a vaccine having polyvalent antigenicity. This strain is being maintained at the Ontario Veterinary College, Dept; of Clinical Studies, University of Guelph, Ontario. Other strains are operative and can be selected for use in certain areas where they are responsiblefor known infections. Mixtures of strains can be used to obtain a degree of polyvalent effect, although the effect is not additive.

The storage of the vaccine must be controlled and ability of the treated bacteria to gradually recover normal growth and activity taken into account. It is not advisable to store the vaccine at room temperatures for longer than about 3 days. Cold temperatures e.g. 4C can prolong the storage life for several weeks or longer. The vaccine has been lyophilized and stored at about 4C for 3 months without significant change.

The vaccine can be administered or injected parentally (e.g. intramuscular, intramammary, subcutaneous Dosage of the vaccine will usually vary from about 1 to about 8 ml at concentrations raging from about 10? to 10 viable bacteria per ml. This corresponds to about 0.02 to about 0.05 g of lyophilized material per ml and'dosages of the order of 1 mg per kg.

of body wt. A 2 ml dose forsows have been found adequate. Upto 20 ml. has'been administered to cows but 4 ml. is usual. The dosage'does not appear to be sharply critical. 'A series of 3 doses'is recommended at intervals of about 1 to 2 weeks between doses, e.g. for sows about 4 weeks, 2 weeks and 1 week before farrowing. Where milk is being drawn from a vaccinated animal,

- the protective effect can be maintained in the milk by doses repeated at intervals. The vaccine was evaluated by vaccinating sows and studying the serologic response, and by feeding colosf'strain of- 0157:KV17 and '08;K87.88a.c. These strains were cultured in Tryptic Soy Broth and incubated for 15 hours at 37C before trum,'milk or serum from vaccinated sows to piglets exposed to infection under controlled conditions. Cows have also been vaccinated and the antibody activity and protective value of their milk evaluated. Good protection in piglets are obtained by feeding them whey from vaccinated milk cows. Milk from vaccinated cows has been spray-dried and reconstituted without apparent loss of protective effect.

The following Examples are illustrative.

': EXAMPLE 1 Two strains of E. coli were selected i.e. the EWl 20 the P307 strain of treatment with various concentrations of formalin. Formalin was added to the 15-hour broth cultures to -give concentrations of from 0.01 to 0.1% v/v in 0.01%

steps, and from 0.1 to 0.5% in 0.1% steps. The incubation in formalin was for the following times: 10, 15. 20 hours. Bacterial counts were thus performed. the number of colonies per ml. being present on the culture plate were counted 24, 48 and 72 hours from the finish of the formalin incubation.

The results are shown in Tables 1 and 2.

Table 1 Number of Colonies of Strain P 307 Cone. Hrs. of Formalin lncuba- Hours Incubation with Fonnalin v/v tion 10 20 0 24 3.0 X 10" 7.1 X 10 6.9 X 10 0.01 (0.004) 48 4.0 X 10 7.7 X 10" 6.8 X 10" 72 3.9 X 10 7.2 X 10" 6.6 X 10" 24 7.9 X 10 6.6 X 10 6.9 X 10 0.02 (0.008) 48 8.1 X 10 7.3 X 10 6.7 X 10 72 8.1 X 10 7.4 X 10' 6.7 X 10 24 3.0 X 10 1.8 X 10 3.5 X 10 0.03 (0.012) 48 4.1 X 10 3.9 X 10' 2.7 X 10 72 4.1 X 10 3.8 X 10' 3.0 X 10 24 0 0 I 0 0.04 (0.016) 48 7.3 X 10 2.1 X 10 7.5 X 10 72 1.4 X 10 4.3 X 10 1.5 X 10 24 0 0 0 0.05 (0.020) 48 1.0 X 10 3.3 X 10 6.3 X 10 72 4.2 X 10 5.4 X 10 1.3 X 10 24 0 0 0 0.07 (0.028) 48 6.2 X 10 0 0 72 2.0 X 10 0 0 24 0 0 0 0.08 (0.032) 48 0 0 -0 72 0 0 0 0.09 (0.036) 0.1 (0.04) 24 0 0 O 0.2 (0.08) 48 0 0 0 0.3 (0.12) 72 0 O 0 0.4 (0.16) 0.5 (0.20)

24 4.2 X 10 3.1 X 10 4.3 X 10" 8.8 X 10 Dist. 48 4.1 X 10 4.1 X 10 4.3 X 10" 8.7 X10 H,O 72 3.7 X 10 5.7 X 10 4.2 X 10 IL/vol formaldehyde concentration in brackets The greatest concentration of formalin allowing 1;. coll to survive was 0.07 0.08%; above this all were dead. Below 0.02% there was virtually no effect. The degree of survival appears directly related to formalin adjusting the concentration of formalin and the time of exposure for each strain. A plate count of zero at 24 hours but a partial recovery in count at 4872 hours inconcentration between 0.02 and 0.08%. Within 0.04 to 40 dicates an upper limit in severity for an acceptable 0.07% there was a gradual effect related to the time of treatment.

Table 2 Number of Colonies of EWl Strain Conc. Hrs. of Formalin lncuba Hours Incubation with Formalin v/v tion 10 20 0 24 9.2 X 10 2.1 10 2.4 X 10" 0.01 (0.004) 48 1.3 X 10" 2.1 X 10 2.5 X 10 72 1.3 X 10" 2.3 X 10 2.5 X 10 24 9.4 X 10 9.5 X 10" 8.2 X 10 0.02 (0.008) 48 1.6 X 10' 9.5 X 10 8.2 X 10 72 1.6 X 10' 8.5 X10 8.5 X 10 24 2.3 X 10" 1.8 X 10" 1.8 X 10 0.03 (0.012) 48 2.5 X 10" 1.9 X 10" 1.8 X 10 72 2.6 X 10 1.9 X 10 1.9 X 10 24 3.0 X 10 2.3 X 10 1.6 X 10 0.04 (0.016) 48 9.2 X10 3.4 X 10 3.5 X 10 72 1.1 X10 3.2 X 10 3.2 X 10 24 0 0 0 0.05 (0.020) 48 2.0 X 10* 11.0 X 10 1.0 X 10' 72 3.9 X 10 2.3 X 10 0 24 0 0 0 0.07 (0.028) 48 0 0 0 72 3.2 X 10 0 0 24 0 0 0 0.08 (0.032) 48 0 0 0 72 0 0 0 All Others 24 0 0 0 0.09 0.5 48 0 0 0 72 0 0 0 24 3.9 X 10 2.5 X 10 3.6 X 10 2.8 X 10' Dist. 48 3.9 x 10" 2.4 x to 3.6 x 10 H 0 72 3.9 X 10 2.4 X 10" 3.5 X 10 3.0 X 10 IL/vol. 5E formaldehyde concentration in brackets EXAMPLE 2 Strain Al of serotype 0149:](91; K88a,c:Hl0 was used to prepare the vaccine. A tryptic soy broth culture containing 7- 8 X 10 viable bacterial/ml. was-incubated for hours at 37C. in the presence offO. 05l- 7r v/v of formalin in a closed container. The formolized cultures contained approximately 7 X' 10 viable bacteria/ml. The vaccine was newly prepared for each vaccination. Colony morphology of the formolized bacteria was determined after plating on blood agar plates.

Effect of 0.04% Formalin on the Organism After the formolized culture was plated on blood agar, bacteria colonies were not visible after 24 hours of incubation at 37C; however, after 48 hours, colonies of E. coli were evident. They were hemolytic and varied considerably in size and differed in varying degrees from the colonies of nonformolized cultures in appearance.

Strain EWl of 0157, and other strains have also been used to prepare the vaccine in a similar manner. The EWl strain in particular has been found more susceptible to the formalin and the viable cells remaining are about 2 log dilutions less than for instance the Al strain.

EXAMPLE 3 One Landr'ace sow andv 1 2 Yorkshire sows between 12 and 18 months of age at the timeof farrowing were used. They were fed a commercial'sow ration. Cmtobiotic pigs were produced and reared by the method described by Alexander et al, Gnotobiotic Pigsz Procurement, Microbial Flora, Serum Proteins and Lymphathic Tissues, Canad. Vet. J., 10, (l969 98 105.

Vaccination f Yaccination was done with a formolized live E. coli vaccine prepared as described in Example 2. Five sows were vaccinated intramuscularly in each side of the neck. Five sows were vaccinated by inoculation ,via the test canal into the lact iferous duct of a single mammary gland. Each sow was vaccinated 3 times: at- 29 l4fand S'daysbefore farrowing l 14 days of gestation); doses. of vaccine were 4, 6, and 8 ml., respectively'jThe same mammary gland in :a given sow was inoculated each time. Three sows. were used as nonvaccinated controls. I

Bacteria Strain Al of serotype 0l49;K9l;K88a,c:Hl0 was used in preparation of the vaccine inoculated in the sows and was used as the inoculum in the oral challenge exposure of the gnotobiotic pigs.

Approximately 1 l. of colostrum was collected from each sow during parturition. Immediately after collection it was centrifuged at 5,000 g and then held at 4C for 4 hours to allow the fat to congeal. The non-fat portion was removed and centrifuged for two hours at 44,000 g at 22C to remove casein. The resultant whey was. stored at 30C. I

" A pooled sample was prepared by mixing e'qual aliquots of colostral whey from each sowwithin a treatment group. The 'pool"was clarifi'd by passing it through a p'refilter, 30,018 and 0.45M membrane'filter pads in series and'then sterilized by passing through a sterile 022p. membrane filter. The eluant was dispensed in 10 ml aliquots-into sterile 'vacutainers. Sterility of each of the wheys was checked by inoculating two blood plates with 0.5 ml-of the filtered whey. The whey obtained from the sows was used in protectection tests against the enteropathogenic effect of E. coli in ten day old gnotobiotic piglets. Each piglet received one aliquot of colostral whey every eight hours for 72 hours. Six hours after the first whey sample was given, the piglets were infected orally with 0.2 ml of broth culture containing approximately 8 X 10 E. coli per ml. The culture was added to the feed. *ln'this series of experiments 10 ml of the basal feed were replaced with 10 ml of sterile whey.

The survival time from the first whey feeding was recorded for each piglet. Observations were made at the same eight hour intervals as feeding took place. Surviving piglets were killed seven days after the first sample application and are recorded as having survived for 168 hours.

' From the results of the present experiment, in Table 3 below, the co'nclusion can be drawn that colostrum from vaccinated sows fed orally to newborn pigs has some protective effect against enteric infections caused by E. coli, whereas colostrum from nonvaccinated, normal sows has no protective value for pigs against experimental exposure'to the organism.

, EXAMPLE 4 E. Coli' serotype 01492K9l; K88aczHl0 was used for broth vaccine production as in Example 2 and infection purposes.

F'ive'sows were. vaccinated intramuscularly, five sows intramammarily and three sows were'left as unvaccinated controls. Each sow' was vaccinated 28 days prior to the expected farrowing date with 4 ml of the formalinized culture, 6 ml were inoculated 14 days later followed by 8 ml five days before the expected farrowing day (114 days of gestation). J'Approximately one liter of milk was collected from each sow seven days afterfarrowing. The milk was processed as described in Example 3 for the colostrum. Fortyml of the feed were replaced with processed milk whey. Survival times, were-recorded. as in Example 3.

All of the piglets developed profuse diarrhea eight to 16 hours after infection; diarrhea continued through the experiments and no differences in severity of diarrhea or rapidity of onset of dehydration were noted between pigsin the different treatment groups.

Table 4 shows the results of the trial in which the pig lets were given- 40 .ml of sows milk whey. Milk from normal non-vaccinated sows. did not prolong the sur vival time of infected gnotobiotic piglets. In this trial milkfrom both intrammary and intramuscularly vaccinated sows afforded an increase in survival time when compared to that of piglets in the groups receiving eithernon-vaccinated sows milk or condensed cow milk.

Results reported in this Example confirm that protection (reduced mortalityyagainst the enteropathogenic effects of E. coli in pigs can be afforded through the antibodies present in milk, even when fed at very low levels when compared with normal milk intake.

EXAMPLE 5 Quantitatively, IgG constitutes an important immu- Table 4 Survival Time of Gnotobiotic Piglets Source Individual Pig Treatment Litter Survival Time Mean Treatment No. (Hours) (Hours) 40 ml of milk whey from sows 72 168 vaccinated via intramammary route 72 I68 78 I68 40 ml of milk whey from sows 72 I52 vaccinated via intramuscular route 72 I68 80 40 40 ml of milk from non-vaccinated 72 I12 sows 72 56 80 40 Control (Condensed cows milk) 78 40 noglobulin in sows milk. Therefore sufficient IgG was TABLE separated from the colostrum of immunized sows to determine its effectiveness in multiplication inhibition, and pig protection tests. The IgG was isolated by a DEAE batch method, and was calculated to have 5 6.86. To complete the tests, g of the pure IgG were isolated.

Multiplication Inhibition (Ml) Tests Vaccination of sows with the living, formalinized E. coli vaccine of the invention has resulted in a marked decrease in their serum bactericidal activity to the vaccine strain. The serum bactericidal activity present in nonvaccinated sows was replaced or masked on vaccination by the development of a MI factor which was distinct from bactericidal antibody in that it was effective in the absence of complement. In the presence of this Ml factor the vacnal strain of E. coli multiplied at a very much reduced rate when compared to that in control absorbed serum. Colostrum from the vaccinated sows contained MI factor whereas colostrum from non-vaccinated sows contained neither effective bactericidal antibody or the Ml factor. The IgG preparation described herein was found to contain Ml factor as is illustrated in Table 5. The test was performed with a solution containing 10 mg/ml of IgG in saline.

Pig Protection Tests Fifteen gnotobiotic pigs were divided into three groups of five. At 5 days of age the following daily treatments were instituted and continued for 3 days:

1. One g of IgG from the colostrum of immunized sows in 90 ml of phosphate buffered saline.

2. One g of gamma globulins present in 90 ml of di- Iuted colostrum from immunized sows.

3. Control 90 ml of phosphate buffered saline.

The treatments, mixed with the regular diet of condensed cow milk, were given in 3 equal doses on each of the 3 treatment days. Observations were made at 8 hourly intervals. at the time of feeding.

A summary of the results is recorded in Table 6.

Significant protection, as indicated by a prolongation of survival time, was afforded by both colostrum from immunized sows and by lgG isolated from that colostrum.

The effect of colostrum and colostral IgG from vaccinated sows on the multiplication of the homologous vaccinal strain.

Survival times of gnotobiotic pigs infected with serotype 0149:1(91 K88aczH I0 Escherichia coli and fed colostrum or colostral IgG from sows.

Mean Survival Time in'Hours Daily Treatment No. of Pigs 1 gram of Gamma Globulin (In colostral whey from immunized sows) 5 1 gram of IgG (Isolated from colostrum from immunized sows) 5 Phosphate Buffer Saline 5 Standard deviation EXAMPLE 6 In field trials, I20 sows have been vaccinated, 33 by the intramuscular and 87 by the intramammary route, 87 non-vaccinated control sows having been kept. The sows were on 2 commercially-operated farms which were chosen because of their having a persistent and high incidence of neonatal diarrhea.

HERD 1: consisted of approximately sows of predominantly Yorkshire type. Pigs in almost all litters developed diarrhea between 2 and 5 weeks of age. Pure cultures of E.c0li serogroup 0138 were isolated on several occasions, and a homologous vaccine was prepared from the P 570 strain of 0138. The sows were 9 listed according to farrowing dates and divided into replicates of 3. Each replicate consisted of a sow vaccinated into a single mammary gland with 2 ml of the vaccine, another vaccinated intramuscularly and a third left unvaccinated.

HERD 2: A herd of l20 hybrid, predominantly Yorkshire type sows. Pigs in all litters developed diarrhea between 3 and 5 weeks of age. Pure cultures of E. coli serogroup 0157 were isolated from several pigs. A heterologous vaccine, prepared from a non-pathogenic (in gnotobiotic pigs), lab variant of E. coli serogroup 08 (P307) was used in this herd. Equal numbers of sows were vaccinated into the mammary gland as were left as unvaccinated controls. Two ml of the vaccine was inoculated into a single mammary gland.

Each farm was visited once a week for vaccination, the sows were vaccinated 3 times, in the periods 24 31, 10 l7 and 3 10 days before farrowing.

Table 7 is a summary of the results obtained.

The practice in each herd was to treat all pigs in the litter if severe diarrhea occured; the results are presented, therefore, as the number of litter treatments necessitated by the onset of diarrhea. Almost four times as many litter treatments were needed in the control sow litters as were needed in litters of vaccinated sows. Both intramuscular and intramammary routes of vaccine administration significantly reduced the number of treatments necessary per weaned litter compared to that needed in litters of non-vaccinated sows (P 0.05). Similarly, diarrhea recurred in litters of nonvaccinated sows on significantly more occasions than in litters of sows of either vaccinated groups (P 0.05).

As can be seen from Table 7, in the first l0 sows on each farm in each treatment group the incidence of diarrhea was lower in the vaccinated than nonvaccinated sow litters. At the end of the trial the incidence had dropped in the non-vaccinated as well as the vaccinated sow progeny. This isthought to be associated with a reduction in the infection load of the farrowing area.

, TABLE 7 Number of Litter treatments Vaccination first 10 litters Last 10 litters HERD No. l None (control) 13 2 intramammary 2 l intramuscular l g l HERD No. 2 None (control) 21 O intramammary 5 0 EXAMPLE 7 Four pregnant cattle of the Jersey breed were used. At the time of calving each cow was approximately 2 years of age. The E. coli strains used for vaccineproduction are designated Al and P307. Strain Al is an enteropathogen of pigs with the serological formula 0l49zK9lz88aczHlO. Strain P307 is a swine enteropathogen with the antigenic formula O8:K87;88ab:H19. The live formalinized E.c0li vaccine was prepared as described in Example 2. Vaccine was introduced into the test cistern of the left hind (LH) and right front (RF) mammary glands via the teat canal. Heifer 2Z was vaccinated with an antigen prepared from the P307 E. coli strain: 4 ml and 6 ml were inoculated into each gland l5 and 5 days before calving respectively. Heifer 4Z was associated with the P307 antigen: 4 ml, 6 ml, 8 ml was inoculated into each gland 42, 32, 22 and '12 days before calving respectively.

Heifer W4 was vaccinated with an antigen prepared from the Al strain of E. call: 4 ml, 6 ml, and 8 ml were inoculated into each gland 26, 16 and 6 days before calving respectively. Heifer 6Z was vaccinated with Al antigen: 4 ml, 6 ml and 5, 8 ml doses were inoculated intoeach gland 95, 85, 75. 65, 55, 45 and 35 days before calving respectively.

Colostrum was taken from each gland on the day that calving occurred and milk from each gland on 2, 3, 4, 7, l4 and 28 days after calving. Further samples were taken from cow 42 on days 42 and 56: this cow was retained until she had calved again 455 days after the last vaccination. Whey was prepared from the colostrum and milk sampled by centrifugation at 44,000 g for 2 hours. The wh'ey was stored in 2 ml aliquots at 30.

The samples taken on days 1 and 2 after calving from cows vaccinated with a vaccine prepared from the Al strain of E. coli, showed an almost complete lack of multiplication when tested against Al organisms. This bacteriostasis was evident in all samples taken up to 4 days after calving in the milk whey from cow 6Z, thereafter bacterial multiplication took place, but to a much greater extent in the whey from non-vaccinated glands than in whey from vaccinated glands. Bacterial multiplication took place in whey from non-vaccinated glands of cow W3 on the third day after calving but was not evident in samples from vaccinated glands until the 14th day after calving. There was an approximate '10- fold difference between the colony count from vaccinated and non-vaccinated gland whey samples from both 6Z and W3 on days 7, l4 and 21 post calving. This difference between vaccinated and non-vaccinated glands was less pronounced, but still significant, in samples taken 'on the 28th day after calving. (P 0.001

They whey samples from cows vaccinated with the P307 strain (4Z and 22) differed in their response in the antibacterial test from the samples from Al vaccinated cows. Bacterial multiplication occurred in colostral whey and in all samples taken thereafter. There was no obvious reductions in the multiplication rate of bacteria in test samples from vaccinated compared to nonvaccinated glands until day 4. This reduction in multiplication rate persisted and was clearly evident in samples taken from vaccinated glands from cow 4Z taken on day 28, but was statistically significant only on days 4, 7 and 14 (P 0.05).

Indirect hemagglutinating antibody titres in milk from vaccinated and non-vaccinated mammary glands The hemagglutinating antibody titres found in milk after intramammary vaccination of 2 of the 4 glands were measured. The titres in colostrum from the inoculated glands (RF and LH) were from 2 to 4 times that found in milk from the non-inoculated glands. The average titre in milk taken from the vaccinated glands 7 days after calving was approximately 7 times that found in milk from the inoculatedglands and by 28 days after calving there was a 14 fold difference in antibody titres between the vaccinated and non-vaccinated gland milk samples. On the 28th day after calving, the average indirect hemagglutinating antibody titre in milk from the non-vaccinated glands was l/50th of that found in colostrum from the same glands, whereas, the titre in milk from vaccinated glands was 1/1 1th of thatin colostrum Protection tests Six day old gnotobiotic pigs, derived from one sow,

were infected with 10* organisms of the Al strain of E. coli and fed 100ml of milk whey 3 times daily for 3 days. The whey was prepared from milk obtained between 7 andl4 days after calving from the vaccinated cows described earlier, and from a non-vaccinated cow. Whey from each of the three sources (Al and P307 vaccinated, and non-vaccinated) was fed to 5 pigs.

EXAMPLE 8 In another trial in progress 4 of 7 pigs being fed milk from non-vaccinated cows died from E. coli infection, while only 1 of 5 being fed reconstituted spraydried milk from cows vaccinated with 0157 strain died, and none of 5 fed reconstituted milk from cows vaccinated with the Al strain of 0149; K91, K88a,c:l-l10. In all cases the pigs were challenged with the Al strain.

Three strains of E. coli from human sources have been used to prepared vaccines according to the invention. These vaccines were used to vaccinate heifers and the milk recovered. It was noticed that calves from the vaccinated heifers did not contract infection whereas calves from twelve contemporary heifers did contract infection and 5 of these 12 died.

EXAMPLE 9 Further field trials have been carried out in four swine herds using the vaccine of the invention. The herds were divided into control (unvaccinated) and vaccinated groups. The mortality of piglets from the control and vaccinated sows was followed. The results are summarized in Table 9. i

TABLE 9 Aver. No. No. alive I No. of Born per at 21 days Herd Litters Litter per litter Survival A Controls 17 10.8 8.0 74% Vaccinated 17 10.8 9.1 84% B Controls 16 10.7 8.4 79% Vaccinated (1M) 25 10.2 8.5 83% C Controls 34 10.7 8.3 77% Vaccinated UM) 33 10.3 8.5 y 83% (lMa) 34 11.1 8.8 79% D Controls 53 10.1 8.0 79% 54 10.5 8.7 83% Vaccinated (lMa) '(IM) intramuscular '(IMa) intramammary Observations were recorded at 8 hour intervals and survival times determined from the time of infection werecompared using the paired t test. The pairs were formed from the treatment within each isolator.

The results of the protection test are presented in Table 8. The survival time of the pigs receiving milk from non-vaccinated cows was significantly shorter than that of pigs in either of the groups being fed milk from vaccinated cows. (P 0.05).

Protection was afforded against the pathogenic effects of the E. coli serogroup Al by milk from homologously vaccinated cows and by milk from cows vaccinated with the heterologous E. coli.

TABLE 8 Survival times (Hours) of gnotohiotic piglets 7 fed milk whcy from vaccinated or non-vaccinated cows lsolator No. of Whey 0 No. pigs Non Vaccinated Vaccinated P307 Al 1 4 62 46.102 1 10 2 4 38 46 54.1 18 3 4 38.62 62 62 4 3 46 94 5 Mean 49 70 x3 6 Standard 1 l 24 26 Deviation Herd A had no diarrhea but diarrhea was present in the other 3 herds. These results show a significant increase in surviving piglets per litter of7c survival where vaccination was carried out even where there was no evidence of E. coli infection in the herd.

l. A bacterial vaccinefor the prevention or treatment of E. coli infections comprising a live strain or strains of E. coli incubated in and modified by dilute solutions of about 0.02 to about 0.08% v/v formalin, to give an altered growth pattern and appearance, and reduced viable cell count.

2. The vaccine of claim 1 wherein the E. coli is selected from the strains EWl of 0157;KV17;P307 of 08;K87.88a,c and A1 of 0149; K91, K88a,c:HlO.

3. The vaccine of claim 1 wherein the viable cell count has been reduced approximately 2 to 7 log dilutions by the formalin.

4. The vaccine of claim 1 in lyophilized form.

5. The vaccine of claim 4 in dosage unit form of about 40 to 400 mg.

6. The vaccine of claim 1 containing broth medium constituents. 

1. A BACTERIA VACCINE FOR THE PREVENTION OR TREATMENT OF E. COLI INFECTIONS COMPRISING A LIVE STRAIN OR STRAINS OF E. COLI INCUBATED IN AND MODIFIED BY DILUTE SOLUTIONS OF ABOUT 0.02 TO ABOUT 0.08% V/V FORMULIN, TO GIVE AN ALTERED GROWTH PATTERN AND APPEARANCE, AND REDUCED VIALBLE CELL COUNT.
 2. The vaccine of claim 1 wherein the E. coli is selected from the strains EWl of 0157;KV17;P307 of 08;K87.88a,c and Al of 0149; K91, K88a,c:H10.
 3. The vaccine of claim 1 wherein the viable cell count has been reduced approximately 2 to 7 log dilutions by the formalin.
 4. The vaccine of claim 1 in lyophilized form.
 5. The vaccine of claim 4 in dosage unit form of about 40 to 400 mg.
 6. The vaccine of claim 1 containing broth medium constituents. 